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United States Patent |
5,326,707
|
Franke
,   et al.
|
July 5, 1994
|
Composition and device for urinary protein assay and method of using the
same
Abstract
A composition, test device and method of determining the presence or
concentration of proteins, such as albumin, in a liquid test sample, such
as urine. The test device includes a test pad with a carrier matrix
incorporating a indicator reagent composition capable of interacting with
proteins to produce a detectable response. The indicator reagent
composition includes an indicator dye that is capable of interacting with
albumin and undergoing a detectable color transition; a buffer; a
hydrophobic polymeric compound having the general structural formula:
##STR1##
wherein A is
##STR2##
and PO is an oxypropylene unit, EO is an oxyethylene unit, y is a number
in the range of 0 to about 20, z is a number in the range of 0 to about
20, the sum y+z is a number in the range of about 2 to about 20, and
R.sub.2 and R.sub.3 are selected, independently, from the group consisting
of hydrogen, an alkyl group, an aralkyl group, and an aryl group;
R.sub.1 is either methylene or oxygen;
n is a number in the range of one to about 8; and
E is hydrogen or methylol when R.sub.1 is methylene, or E is hydroxy when
R.sub.1 is oxygen; and a suitable carrier which is water. The indicator
reagent composition provides an improved color resolution in the test
samples raving negative protein and trace amount of protein.
Inventors:
|
Franke; Gunter (Leichlingen, DE);
Salvati; Michael (St. Paul, MN);
Sommer; Ronald G. (Elkhart, IN)
|
Assignee:
|
Miles Inc. (Elkhart, IN)
|
Appl. No.:
|
800272 |
Filed:
|
November 29, 1991 |
Current U.S. Class: |
436/86; 252/408.1; 422/56; 436/87; 436/88 |
Intern'l Class: |
G01N 033/00 |
Field of Search: |
436/86,87,88
422/55,56,57,408.1
252/408.1
|
References Cited
U.S. Patent Documents
4013416 | Mar., 1977 | Rittersdorf et al. | 436/86.
|
5013527 | May., 1991 | Arai et al. | 422/58.
|
5049358 | Sep., 1991 | Lau | 436/86.
|
5096833 | Mar., 1992 | Lau et al. | 436/86.
|
5124266 | Jun., 1992 | Coryn et al. | 436/88.
|
5187104 | Feb., 1993 | Corey et al. | 436/86.
|
Primary Examiner: Housel; James C.
Assistant Examiner: Tran; Lien
Attorney, Agent or Firm: Coe; Roger N.
Claims
What is claimed is:
1. An indicator reagent composition capable of exhibiting a sufficient
color transition upon contacting a protein-containing test sample to
demonstrate the presence or concentration of protein in the test sample
consisting essentially of:
(a) an indicator dye capable of interacting with a protein and exhibiting a
color transition upon such interaction, said indicator dye being present
in an amount of about 0.05 to about 0.6 millimoles per liter of the
composition and wherein the indicator dye is selected from the group
consisting of 3',3",5,5"-tetraiodo-3,4,5,6-tetrabromophenolsulfophthalein,
3,3"-diiodo-5,5",3,4,5,6-hexabromophenolsulfophthalein, and combinations
thereof;
(b) a buffer present in an amount of about 250 to about 750 millimoles per
liter of the composition wherein the buffer is selected from the group
consisting of citric acid, maleic acid, tartaric acid, phthalic acid,
sulfosalicyclic acid, succinic acid, malonic acid, their respective alkali
metal and ammonium salts and combinations thereof;
(c) a hydrophobic polymeric compound present in an amount of about 1% to
about 8% by weight per milliliter of the composition having the general
structural formula:
H--[--A--R.sub.1 --].sub.n --A--E,
wherein A is
##STR18##
and PO is an oxypropylene unit, EO is an oxyethylene unit, y is a number
in the range of 0 to about 20, z is a number in the range of 0 to about
20, the sum of y+z is a number in the range of about 2 to about 20, and
R.sub.2 and R.sub.3 are selected, independently, from the group consisting
of hydrogen, alkyl group, an aralkyl group, and an aryl group;
R.sub.1 is methylene or oxygen;
n is a number in the range of 1 to about 8; and
E is hydrogen or methylol when R.sub.1 is methylene, or E is hydroxy when
R.sub.1 is oxygen; and
(d) a carrier vehicle for said composition.
2. The composition of claim 1 wherein the buffer buffers the composition at
a pH of 2 to 4.
3. The composition of claim 1 wherein the hydrophobic polymeric compound is
present in the amount of about 2% to about 6%, by weight, per milliliter
of the composition.
4. The composition of claim 1 wherein the hydrophobic polymeric compound
has a molecular weight in the range of about 800 to about 12,000.
5. The composition of claim 1 wherein the substituents R.sub.2 and R.sub.3
of the hydrophobic polymeric compound are selected, independently, from
the group consisting of hydrogen, an alkyl group including from one to
about 22 carbon atoms, .alpha.-methylstyryl, and phenyl.
6. The composition of claim 1 wherein the moiety --A--R.sub.1 --of the
hydrophobic polymeric compound is
##STR19##
wherein R.sub.2 ' and R.sub.3 ' are, independently, hydrogen or an alkyl
group including one to about 22 carbon atoms.
7. The composition of claim 1 wherein n is a number in the range of about 2
to about 5.
8. The composition of claim 1 wherein the moiety --A--R.sub.1 --of the
hydrophobic polymeric compound is
##STR20##
wherein PO is an oxypropylene unit, EO is an oxyethylene unit, y' and z'
are, independently, numbers in the range of about 2 to about 8, the sum
y'+z' is a number in the range of about 6 to about 16; and R.sub.2 ' is an
alkyl group including from about 6 to about 18 carbon atoms.
9. The composition of claim 8 wherein y' and z' are, independently, numbers
in the range of about 5 to about 6; the sum y'+z' is a number in the range
of about 10 to about 12; and R.sub.2 ' is an alkyl group including from
about 7 to about 12 carbon atoms.
10. The composition of claim 1 having a buffered pH in the range of about 2
to about 4.
11. The method of contacting a test sample with an indicator reagent
composition to determine the presence of concentration of protein in the
test sample, said method being essentially free of interferences
attributed to specific gravity, said method comprising the steps of:
(i) contacting the test sample with an indicator reagent composition
consisting essentially of:
(a) an indicator dye capable of interacting with a protein and exhibiting a
color transition upon such interaction, said indicator dye being present
in an amount of about 0.05 to about 0.6 millimoles per liter of the
composition and wherein the indicator dye is selected from the group
consisting of 3',3",5,5"-tetraiodo-3,4,5,6-tetrabromophenolsulfophthalein,
3,3"-diiodo-5,5",3,4,5,6-hexabromophenolsulfophthalein, and combinations
thereof;
(b) a buffer present in an amount of about 250 to about 750 millimoles per
liter of the composition wherein the buffer is selected from the group
consisting of citric acid, maleic acid, tartaric acid, phthalic acid,
sulfosalicylic acid, succinic acid, malonic acid, their respective alkali
metal and ammonium salts and combinations thereof;
(c) a hydrophobic polymeric compound present in an amount of about 1% to
about 8% by weight per milliliter of the composition having the general
structural formula:
H--[--A--R.sub.1 --].sub.n --A--E,
wherein A is
##STR21##
and PO is an oxypropylene unit, EO is an oxyethylene unit, y is a number
in the range of 0 to about 20, z is a number in the range of 0 to about
20, the sum of y+z is a number in the range of about 2 to about 20, and
R.sub.2 and R.sub.3 are selected, independently, from the group consisting
of hydrogen, alkyl group, an aralkyl group, and an aryl group;
R.sub.1 is methylene or oxygen;
n is a number in the range of 1 to about 8; and
E is hydrogen or methylol when R.sub.1 is methylene, or E is hydroxy when
R.sub.1 is oxygen; and
(d) a carrier vehicle for said composition; and
(ii) determining the presence or concentration of protein in the test
sample from a resulting intensity or degree of the color transition of the
indicator reagent composition.
12. The method of claim 11 wherein the intensity and degree of the color
transition is determined visually or by instrument.
13. The method of claim 11 wherein the presence or concentration of protein
is determined by a dry phase assay.
14. The method of claim 11 wherein the test sample is a biological sample.
15. The method of claim 14 wherein the biological sample is urine, blood
plasma or blood serum.
16. The method of claim 15 wherein the urine has a specific gravity of
about 1.005 to about 1.030.
17. The method of claim 11 wherein the test sample includes from 0 mg/dL
protein to about 30 mg/dL protein.
18. The method of claim 11 wherein the buffer buffers the indicator reagent
composition at a pH of 2 to 4.
19. The method of claim 11 wherein the number n of the hydrophobic
polymeric compound is in the range of about 2 to about 5.
20. The method of claim 11 wherein the moiety --A--R.sub.1 --of the
hydrophobic polymeric compound is
##STR22##
wherein R.sub.2 ' and R.sub.3 ' are, independently, hydrogen or an alkyl
group including one to about 22 carbon atoms.
21. The method of claim 20 wherein n is a number in the range of about 2 to
about 5.
22. The method of claim 11 wherein the moiety --A--R.sub.1 --of the
hydrophobic polymeric compound is
##STR23##
wherein PO is an oxypropylene unit, EO is an oxyethylene unit, y' and z'
are, independently, numbers in the range of about 2 to about 8, the sum
y'+z' is a number in the range of about 6 to about 16; and R.sub.2 ' is an
alkyl group including from about 6 to about 18 carbon atoms.
23. The method of claim 22 wherein y' and z' are, independently, numbers in
the range of about 5 to about 6; the sum y'+z' is a number in the range of
about 10 to about 12; and R.sub.2 ' is an alkyl group including from about
7 to about 12 carbon atoms.
24. The method of claim 11 wherein the liquid test sample is urine.
25. The method of claim 11 wherein the liquid test sample contains about 30
mg/dL or less of protein.
26. The method of claim 11 wherein the liquid test sample contains about 15
mg/dL or less of protein.
27. The method of claim 11 wherein the liquid test sample is urine having a
specific gravity of about 1.005 to about 1.030.
28. An analyte detection device for contacting with a liquid test sample to
determine the presence or concentration of protein in the liquid test
sample, comprising:
(i) a support strip; and
(ii) a test pad comprising a carrier matrix having an indicator reagent
composition incorporated therein, said indicator reagent composition
consisting essentially of:
(a) an indicator dye capable of interacting with a protein and exhibiting a
color transition upon such interaction, said indicator dye being present
in an amount of about 0.05 to about 0.6 millimoles per liter of the
composition and wherein the indicator dye is selected from the group
consisting of 3',3",5,5"-tetraiodo-3,4,5,6-tetrabromophenolsulfophthalein,
3,3"-diiodo-5,5",3,4,5,6-hexabromophenolsulfophthalein, and combinations
thereof;
(b) a buffer in an amount of about 250 to about 750 millimoles per liter of
the composition wherein the buffer is selected from the group consisting
of citric acid, maleic acid, tartaric acid, phthalic acid, sulfosalicyclic
acid, succinic acid, malonic acid, their respective alkali metal and
ammonium salts and combinations thereof;
(c) a hydrophobic polymeric compound present in an amount of about 1% to
about 8% by weight per milliliter of the composition having the general
structural formula:
H--[--A--R.sub.1 --].sub.n --A--E,
wherein A is
##STR24##
and PO is an oxypropylene unit, EO is an oxyethylene unit, y is a number
in the range of 0 to about 20, z is a number in the range of 0 to about
20, the sum of y+z is a number in the range of about 2 to about 20, and
R.sub.2 and R.sub.3 are selected, independently, from the group consisting
of hydrogen, alkyl group, an aralkyl group, and an aryl group;
R.sub.1 is methylene or oxygen;
n is a number in the range of 1 to about 8; and
E is hydrogen or methylol when R.sub.1 is methylene, or E is hydroxy when
R.sub.1 is oxygen; and
(d) a carrier vehicle for said composition.
Description
FIELD OF THE INVENTION
The present invention relates to an improved composition, assay device and
method of assaying a test sample for the presence or concentration of
protein. More particularly, the present invention relates to a
composition, method and device for assaying a liquid test sample, such as
urine, for proteins, and especially for negative and trace amounts of
proteins, by utilizing a test device including a test pad comprising a new
and improved indicator reagent composition incorporated into a carrier
matrix, wherein a detectable or measurable response occurs upon contact of
the test pad with a protein-containing liquid. The new and improved
indicator reagent composition comprises: (1) an indicator dye that is
capable of interacting with albumin and undergoing a detectable response,
such as a color transition; (2) a buffer; and (3) a hydrophobic polymeric
compound having the general structural formula (I):
##STR3##
wherein A is
##STR4##
and PO is an oxypropylene unit, EO is an oxyethylene unit, y is a number
in the range of 0 to about 20, z is a number in the range of 0 to about
20, the sum y+z is a number in the range of about 2 to about 20, and
R.sub.2 and R.sub.3 are selected, independently, from the group consisting
of hydrogen, an alkyl group, an aralkyl group, and an aryl group;
R.sub.1 is either methylene or oxygen;
n is a number in the range of one to about 8; and
E is hydrogen or methylol when R.sub.1 is methylene, or E is hydroxy when
R.sub.1 is oxygen.
The new indicator reagent composition provides an improved color resolution
in the test pad, and therefore an improved color differentiation between
test samples assayed as including a negative amount of protein and test
samples assayed as including a trace amount of protein. Accordingly, the
number of false positive assays for protein is substantially reduced. By
substantially reducing the number of false positive assays for albumin,
fewer unnecessary confirmatory assays to verify the presence of a trace
amount of protein in a test sample are performed. In addition, the present
invention relates to incorporating the new indicator reagent composition
into a carrier matrix to provide a test pad of a test device in an
improved method to determine the presence or concentration of proteins,
like albumin, in a test sample by a dry phase, test strip assay procedure.
BACKGROUND OF THE INVENTION AND PRIOR ART
Albumin is the most abundant plasma protein, generally constituting
slightly over one-half of the total protein in mammalian plasma. In the
human body, albumin has the important role of regulating the water balance
between blood and tissues, and of functioning as a transport molecule for
various compounds, such as bilirubin, fatty acids, cortisol, thyroxine and
drugs like sulfonamides and barbiturates, that are only sparsely soluble
in water. An albumin deficiency can restrict the transport of sparsely
water soluble materials throughout the body and a deficiency is signaled
in an individual by an abnormal accumulation of serous fluid, or edema.
Therefore, it is clinically important to determine whether an individual
has a deficiency of serum albumin.
Likewise, it is clinically important to determine if an individual is
excreting an excess amount of protein. A normal functioning kidney forms
urine in an essentially two step process. Blood flows through the
glomerulus, or glomerular region of the kidney. The capillary walls of the
glomerulus are highly permeable to water and low molecular weight
components of the blood plasma. Albumin and other high molecular weight
proteins cannot pass through these capillary walls and essentially are
filtered out of the urine so that the protein is available for use by the
body. The liquid containing the low molecular weight components passes
into the tubules, or tubular region, of the kidney where reabsorption of
some urine components, such as low molecular weight proteins; secretion of
other urine components; and concentration of the urine occurs. AM a
result, through the combined processes of the glomerulus and tubules, the
concentration of proteins in urine should be minimal. Therefore,
abnormally high amounts of albumin in urine must be detected and related
to a physiological dysfunction.
A relatively high concentration of albumin in the urine of an individual
usually is indicative of a diseased condition. For example, the average
normal concentration of protein in urine varies from about 10 mg/dL
(milligrams per deciliter) to about 20 mg/dL, with approximately one-fifth
of the total urinary protein being serum albumin. However, in a majority
of diseased states, urinary protein levels increase appreciably, such that
albumin accounts for from about 60 percent to about 90 percent of the
excreted protein. The presence of an abnormal increased amount of protein
in the urine, known as proteinuria, is one of the most significant
indicators of renal disease, and may be indicative of various other
non-renal related diseases.
Therefore, in order to determine if an individual has an albumin deficiency
or to determine if an individual excretes an excess amount of protein, and
in order to monitor the course of medical treatment to determine the
effectiveness of the treatment, simple, accurate and inexpensive protein
detection assays have been developed. Furthermore, of the several
different assay methods developed for the detection or measurement of
protein in urine and serum, the methods based on dye binding techniques
have proven especially useful because dye binding methods are readily
automated and provide reproducible and accurate results.
In general, dye binding techniques utilize pH indicator dyes that are
capable of interacting with a protein, such as albumin, and that are
capable of changing color upon interaction with a protein absent any
change in pH. When a pH indicator dye interacts with, or binds to, a
protein, the apparent pK.sub.a (acid dissociation constant) of the
indicator dye is altered and the dye undergoes a color transition,
producing the so-called "protein-error" phenomenon. In methods utilizing
the dye binding technique, an appropriate buffer maintains the pH
indicator dye at a constant pH to prevent a color transition of the pH
indicator dye due to a substantial shift in pH. Due to the "protein-error"
phenomena, the pH indicator dye undergoes a color transition upon
interaction with protein that is identical to the color change arising
because of a change in the pH. Examples of pH indicator dyes used in the
dry phase assay of proteins that are capable of interacting with or
binding to proteins and exhibiting "protein-error" color transitions
include tetrabromophenol blue (TBPB) and
tetrachlorophenol-3,4,5,6-tetrabromosulfophthalein.
Although pH indicator dyes have been used extensively in protein assays,
several disadvantages still exist in protein assay methods utilizing
indicator dyes. For example, methods based upon pH indicator dyes cannot
sufficiently differentiate, quantitatively, between a trace protein
concentration of about 15 to about 30 mg/dL and a negative protein
concentration below about 15 mg/dL. A negative protein concentration is
the normal background amount of protein present in urine, and is
clinically insignificant. A trace protein concentration shows a slightly
elevated amount of protein in urine and is clinically significant. An
assay showing a trace amount of protein requires a confirmatory assay to
conclusively show that an elevated amount of protein is present in the
urine. Although several simple quantitative assays are available for the
determination of the total protein content in a test sample, the majority
of these assay methods, with the notable exception of the simple
colorimetric reagent test strip, require the precipitation of protein to
make quantitative protein determinations. Accordingly, the confirmatory
assays are more time consuming and expensive than the test strip assays
used to screen the urine samples for protein content. Therefore, a need
exists for a test strip assay that substantially reduces the number of
false positive assays for a trace amount of protein.
The colorimetric reagent test strip utilizes the previously discussed
ability of proteins to interact with certain acid-base indicators and to
alter the color of the indicator without any change in the pH. For
example, when the indicator tetrabromophenol blue (TBPB) is buffered to
maintain a constant pH of approximately 3, the test pad remains a yellow
color upon contact with a test sample that does not contain protein.
However, for test samples containing protein, the presence of protein
causes the buffered dye to impart either a greenish-yellow color, a green
color or a blue color to the test pad, depending upon the concentration of
protein in the test sample. Consequently, the development of a
greenish-yellow color in the test pad of a dry phase test strip can be
interpreted as a trace amount of protein or as a negative amount of
protein.
Some colorimetric test strips used in protein assays have a single test
area consisting of a small square pad of a carrier matrix impregnated with
a buffered pH indicator dye, such as tetrabromophenol blue. Other
colorimetric test strips are multideterminant reagent strips that include
one test area, or test pad, for the protein assay as described above, and
further include several additional test pads on the same strip to permit
the simultaneous assay of other urinary constituents, like pH. For both
types of colorimetric test strips, the assay for protein in urine is
performed simply by dipping the colorimetric test strip into a well mixed,
uncentrifuged urine sample, then comparing the resulting color of the test
pad of the test strip to a standardized color chart provided on the
colorimetric test strip bottle.
For test strips utilizing tetrabromophenol blue, buffered at pH 3, as the
indicator dye, quantitative assays for protein can be performed and are
reported as negative, trace, or one "plus" to four "plus". A negative
reading, or yellow color, indicates that the urine contains less than
about 15 mg/dL protein, as demonstrated by the lack of a color transition
of the indicator dye. A trace reading, or greenish-yellow color, indicates
that the urine contains from about 15 to about 30 mg/dL of protein. The
one "plus" to four "plus" readings, signified by color transitions of
green through increasingly dark shades of blue, are approximately
equivalent to urine protein concentrations of 30, 100, 300, and over 2000
mg/dL protein, respectively, and serve as reliable indicators of
increasingly severe proteinuria. Therefore, differentiating between a
negative assay (yellow color) and a trace assay (greenish-yellow color) is
important for an accurate protein analysis.
In accordance with the above-described method, an individual can readily
determine, visually, that the protein content of a urine sample is in the
range of 0 mg/dL to about 30 mg/dL. However, the color differentiation
afforded by the presently available commercial test strips is insufficient
to allow an accurate determination of urinary protein content between a
sample having less than about 15 mg/dL protein (negative) and a sample
including from about 15 to about 30 mg/dL protein (trace). The inability
to differentiate between low urinary protein concentrations is important
clinically because a healthy person usually has a urine protein level in
the range of about 2 mg/dL to about 20 mg/dL. Therefore, it is clinically
important to determine precisely the urine protein content of an
individual, rather than merely estimating the protein content at some
value less than about 30 mg/dL.
A trace reading for urinary protein is considered a positive assay, and
confirmation of a positive test strip reading is required. The prevalent
method of confirming a test strip positive protein assay is the
turbidimetric sulfosalicylic acid method, abbreviated as SSA. A high
frequency of false positive assays, requires confirmatory testing for each
false positive assay, and the attendant added cost. Therefore, it is
important that a screening test for protein, like a test strip assay,
provide a low frequency of false positive readings.
Trace proteinuria is defined as protein excretion slightly above normal
proteinuria. Normal excretion of protein is 50-150 mg/24 hours and 200-300
mg/24 hours in pregnancy. Using an average urine volume of 1250 mL/24 hrs,
concentration units of 4-12 mg/dL (16-20 mg/dL in pregnancy) are
calculated. Since 24 hour urine volumes vary from about 700 mL to more
than 2000 mL, the range of normal protein is considerably wider, and, as
expected, the more concentrated urine samples of higher specific gravity
(SG) contain more protein. Trace proteinuria, then, is the concentration
of protein that falls between negative and one "plus" (30 mg/dL). However,
since normal proteinuria covers a range of protein concentrations, trace
proteinuria also covers a range of protein concentrations. The trace
protein concentration also is dependent on the specific gravity, or SG, of
the sample.
The variation of protein concentration with SG does not effect the
confirmatory SSA assay, and therefor protein precipitation in the SSA
assay is considered indicative of clinical proteinuria. However, the SSA
method also has limitations because it is a qualitative method and because
procedures vary between clinical laboratories. Accordingly, the problem
with a trace reading for protein provided by a screening test, such as a
dry phase test strip, is that the reading must correspond to a protein
range that is not well defined for clinical samples; that is dependent on
the SG of the sample; and that overlaps with the normal, or negative,
protein range. Further, trace readings are usually confirmed as positive
by a qualitative method that has not been standardized, but is the method
of choice in the art because the method is easy, requires no
instrumentation, and detects clinical proteinuria in the presence of
normal protein.
Of course, the protein content of a urine sample can be determined more
precisely by quantitative 24 hour protein precipitation techniques.
However, these tests are time consuming and relatively expensive.
Furthermore, the precipitation tests must be run in a laboratory by
trained personnel, and therefore are unavailable for the patient to
perform at home to quickly determine urine protein content and to monitor
the success or failure of a particular medical treatment.
Therefore, it would be extremely advantageous to have a simple, accurate
and trustworthy method of assaying urine for protein content that allows
visual differentiation of protein levels in the ranges of 0 mg/dL to about
15 mg/dL and about 15 mg/dL to about 30 mg/dL, and upwards to between
about 100 mg/dL to about 300 mg/dL. By providing such an accurate method
of determining urine protein concentration in an easy to use form, such as
a dip-and-read test strip, the urine assay can be performed by laboratory
personnel to afford immediate test results, such that a diagnosis can be
made without having to wait up to one day for assay results and medical
treatment can be commenced immediately. In addition, the test strip method
can be performed by the patient at home to more precisely monitor low
levels of protein in urine and monitor the success of the medical
treatment the patient is undergoing, without providing a large number of
false positive assays that require unnecessary, time consuming and costly
confirmatory testing. Finally, the method and composition used in a
protein assay should be independent of the specific gravity of the urine
to provide an accurate protein assay.
For example, the current urinary protein reagent test strips contain an
octahalosulfophthalein protein indicator, e.g., tetrabromophenol blue
(TBPB), as the indicator dye. When these strips are dipped into
albumin-free urine samples of low to medium specific gravity, e.g., SG
less than 1,020, the strips turn to a yellowish-green color. When the same
strips are dipped into an albumin-free, high SG urine sample, e.g., SG
equal to or greater than 1,020, the strips turn to a greenish-yellow
color. This greenish-yellow color easily can be interpreted incorrectly as
a clinically significant trace concentration of albumin (10-15 mg/dL).
However, even with low SG urine samples, the negative color is difficult
to differentiate from a true trace color.
As will be described more fully hereinafter, the method of the present
invention allows the fast, accurate and trustworthy protein assay of urine
by utilizing a test strip that includes a test pad comprising a carrier
matrix incorporating a new and improved indicator reagent composition.
Surprisingly and unexpectedly, the indicator reagent composition of the
present invention essentially eliminates the interfering effects of
specific gravity on the assay of urine samples including negative to trace
amounts of protein. The new and improved indicator reagent composition of
the present invention enhances visual color resolution by essentially
eliminating the development of an interfering greenish-yellow color by
high SG test samples including a negative amount of albumin. Therefore the
sensitivity of the assay is enhanced, allowing urine protein
concentrations to be determined accurately at levels of about 30 mg/dL or
less, and precluding costly confirmatory testing arising from a false
positive screening assay for albumin. In addition, the method of the
present invention also can be used to determine the presence or
concentration of higher concentrations of proteins, such as from about 100
mg/dL to about 2000 mg/dL, in a test sample.
Proteinuria resulting from abnormally high albumin levels depends upon the
precise nature of the clinical and pathological disorder and upon the
severity of the specific disease. Proteinuria can be intermittent or
continuous, with transient, intermittent proteinuria usually being caused
by physiological or functional conditions rather than by renal disorders.
Therefore, accurate assays of urine and other test samples for protein
must be available for both laboratory and home use. The assays must permit
the detection or measurement of the proteins of interest, such that a
correct diagnosis can be made and correct medical treatment implemented,
monitored and maintained. In addition, it would be advantageous if the
protein assay method could be utilized in a dip-and-read format for the
easy and economical, qualitative or quantitative determination of protein
in urine or other test samples.
Furthermore, any method of assaying for protein in urine or other test
samples must yield accurate, trustworthy and reproducible results by
utilizing a method that provides a detectable or measurable color
transition as a result of an interaction between the indicator reagent
composition and the protein, and not as a result of a competing chemical
or physical interaction, such as a pH change or preferential interaction
with a test sample component other than protein. Moreover, it would be
advantageous if the protein assay method is suitable for use in dry
reagent strips for the rapid, economical and accurate determination of
protein in urine and other test samples. Additionally, the method and test
pad, comprising the carrier matrix and the indicator reagent composition,
utilized in the assay for protein, and the new indicator reagent
composition, should not adversely affect or interfere with the other test
reagent pads that are present on multideterminant test pad strips.
Although a dry phase chemistry test strip utilizing a dye, such as
tetrabromophenol blue or
tetrachlorophenol-3,4,5,6-tetrabromosulfophthalein, has been used
extensively for several years, no dry phase test strip has utilized a test
pad comprising a carrier matrix, such as a filter paper, homogeneously
impregnated with an indicator reagent composition including a hydrophobic
polymeric compound as depicted above in general structural formula (I).
The indicator reagent composition responds to urinary proteins and is
essentially independent of urine specific gravity, thereby essentially
eliminating the development of an interfering greenish-yellow color in the
test pad by high SG samples including a negative amount of protein.
Therefore, the assay exhibits an improved visual color resolution and an
increased assay sensitivity, especially at lower protein concentration
levels, to substantially reduce the number of false position assays.
Surprisingly and unexpectedly, because of the essential elimination of the
interferences related to urine specific gravity, the method of the present
invention facilitates the dry phase test strip assay of urine and other
test sample for albumin, especially at albumin levels of 0 mg/dL to about
30 mg/dL.
The prior art contains numerous references relating to the wet phase and
the dry phase chemistry utilized in the pH indicator dye method of
assaying urine for proteins. For example, Keston U.S. Pat. No. 3,485,587
discloses the basic dye binding technique used to assay for proteins at a
constant pH. Keston teaches utilizing a single indicator dye, maintained
at a constant pH slightly below the pK.sub.a (acid dissociation constant)
of the dye and impregnated into a dry test paper, like filter paper, to
determine the presence or concentration of albumin by monitoring the color
transition of the dye. Free et al., in U.S. Pat. No. 3,095,277, also
disclose a method of detecting the albumin content of liquid test samples
by incorporating a suitable indicator composition into a bibulous carrier,
like untreated filter paper. Similarly, Atkinson et al., in U.S. Pat. No.
3,438,737, disclose a test device comprising a test composition
impregnated into an untreated bibulous matrix, such as filter paper, wood
strips, synthetic plastic fibrous materials, nonwoven fabrics and woven
fabrics for detecting protein in fluids.
Rittersdorf et al., in U.S. Pat. No. 4,013,416, disclose a test strip
wherein an absorbent carrier is impregnated with an octahalosulfophthalein
pH indicator dye, a buffer and a water-insoluble polypropylene glycol
having a molecular weight of from about 500 to about 10,000 daltons.
Rittersdorf et al. teach that the water-insoluble polypropylene glycol
reduces the reactivity of the indicator dye with nitrogen containing
compounds, such as metabolites of pharmaceuticals, thereby reducing the
blank reaction in test strips. Rittersdorf et al. also teach only that
water-insoluble propylene glycols are useful, e.g. polyethylene glycols
are not useful. Rittersdorf et al. do not teach or suggest the usefulness
of a polymer including a hydrocarbon, or essentially a hydrocarbon,
backbone including pendant polyoxyalkylene units. In contrast, the
indicator reagent composition of the present invention includes a
hydrophobic polymeric compound having a hydrocarbon, or essentially
hydrocarbon, backbone including 1 to about 8 alkylphenol units, like
nonylphenol, linked by a methylene group or oxygen group, wherein the
phenol moiety of each alkylphenol unit is ethoxylated and/or propoxylated
to include about 2 and up to about 20 ethoxy and/or propoxy units in
total.
The above-cited references do not teach or suggest, either alone or in
combination, that an indicator reagent composition including a hydrophobic
polymeric compound, as depicted above in general structural formula (I),
can be used in a diagnostic device to achieve a more accurate
determination of the amount of an analyte, like protein, and especially
low amounts of an analyte, in a test sample. The references also do not
teach or suggest, alone or in combination, that such an indicator reagent
composition substantially reduces the number of false positive assays for
albumin by essentially eliminating the effects of urine specific gravity
in the assay for urinary proteins.
In contrast to the prior art, and in contrast to the presently available
commercial test strips, the method of the present invention provides
increased sensitivity in the detection and measurement of proteins in a
liquid test sample, such as a biological fluid, like urine. Surprisingly
and unexpectedly, by utilizing an indicator reagent composition,
comprising an indicator dye, a buffer and a hydrophobic polymeric compound
depicted by general structural formula (I), homogeneously impregnated into
a carrier matrix, an assay of a test sample including a negative amount of
protein (e.g., less than about 15 mg/dL) can be differentiated from an
assay of a test sample including a trace amount of protein (e.g., about 15
to about 30 mg/dL) more accurately. Accordingly, the number of false
positive assays is reduced substantially, and the number of unnecessary
and costly confirmatory assays also is reduced. Hence, in accordance with
the method of the present invention, new and unexpected results are
achieved in the dry phase test strip assay of urine and other test samples
for proteins by utilizing a test pad, comprising a carrier matrix having
homogeneously incorporated therein an indicator reagent composition
comprising an indicator dye, a buffer and a hydrophobic polymeric compound
of general structural formula (I), that provides an accurate protein assay
for samples including a negative to low trace amount of protein, and that
is independent of the specific gravity of the test sample.
SUMMARY OF THE INVENTION
In brief, the present invention is directed to a new and improved method,
test device and composition for determining the presence or concentration
of a component in a test sample, especially negative to trace amounts of
the component. The method includes using an indicator reagent composition
capable of interacting with a test sample component to produce a
detectable response. For home use, the indicator reagent composition
produces a visually detectable response. For laboratory use, the indicator
reagent composition produces a response that is detectable visually or by
instrument. The method is suitable for a dry phase assay wherein the
indicator reagent composition is incorporated into a carrier matrix of an
analyte detection device. The carrier matrix of the analyte detection
device comprises a bibulous porous material, such as filter paper, or a
nonbibulous porous material, such as a permeable strip, layer or membrane
of a polymeric material. An indicator reagent composition is homogeneously
incorporated into the carrier matrix, and the carrier matrix then holds
the indicator reagent composition homogeneously throughout the carrier
matrix in a known concentration while maintaining carrier matrix
penetrability for the liquid test sample.
More particularly, the present invention is directed to a method of
assaying urine or other test samples for proteins, especially negative to
trace quantities of proteins, by utilizing a new and improved indicator
reagent composition. It has been demonstrated that employing an indicator
reagent composition including an indicator dye, a buffer and a hydrophobic
polymeric compound of general structural formula (I) affords sufficiently
increased sensitivity and sufficient color resolution at low protein
concentrations to permit the differentiation between a negative amount of
protein, e.g., less than about 15 mg/dL, and a trace amount of protein,
e.g., from about 15 to about 30 mg/dL, in a liquid test sample. The assay
results are essentially independent of the specific gravity of the test
sample. In accordance with an important feature of the present invention,
the qualitative or quantitative determination of protein levels between 0
mg/dL and about 2000 mg/dL, and especially between 0 mg/dL and about 30
mg/dL, in urine and other test samples is accomplished.
By utilizing the indicator reagent composition of the present invention in
clinical test methods, the qualitative or quantitative concentration of
proteins, such as albumin, in urine or other test samples can be
accurately determined, especially at negative to trace concentrations of
protein, because the response of the indicator reagent composition is
independent of the specific gravity of the test sample. Surprisingly and
unexpectedly, the indicator reagent composition incorporated into the
analyte detection device allows the differentiation between a negative
protein concentration and between a trace protein concentration in urine
and other test samples having a specific gravity in the range of 1.005 to
about 1,030, thereby significantly reducing the number of false positive
assays for protein in urine.
Therefore, one aspect of the present invention is to provide a new and
improved test device, method and composition for determining the relative
concentration of a chemical compound in a liquid. Another aspect of the
present invention is to provide a simple, accurate and reproducible method
of assaying urine or other test samples for proteins, especially at
protein concentrations of 30 mg/dL and less.
Another aspect of the present invention is to provide a new and improved
protein interactive test device for interaction with protein in a test
fluid to produce a visible change, such as a change in color, of the test
device, indicative of the protein concentration in the test fluid.
Another aspect of the present invention is to provide a method of assaying
urine or other liquid test samples having sufficient sensitivity and
sufficient visual color resolution to allow the differentiation between
negative and trace protein concentrations.
Another important aspect of the present invention is to provide a method of
assaying urine or other liquid test samples that is sensitive to protein
concentrations of about 30 mg/dL and less, and that quantitatively
discriminates between protein levels of 0 mg/dL to about 2000 mg/dL, and
especially 0 mg/dL to about 30 mg/dL.
Another aspect of the present invention is to provide a method of assaying
urine or other test liquids utilizing an indicator reagent composition
that can interact with proteins and undergo a detectable and measurable
color transition to establish the presence and concentration of protein in
the test sample.
Another aspect of the present invention is to provide an indicator reagent
composition that can interact with proteins and undergo a visually or
instrumentally differentiable color transition to allow the quantitative
determination of the concentration of protein in the urine or other liquid
samples at levels between 0 mg/dL and about 2000 mg/dL, and especially
between 0 mg/dL and about 30 mg/dL.
Another aspect of the present invention is to provide an indicator reagent
composition that is capable of interacting with albumin and undergoing a
color change, said indicator reagent composition comprising an (a)
indicator dye; (b) a buffer; (c) a hydrophobic polymeric compound having
the general structural formula (I):
##STR5##
wherein A is
##STR6##
and PO is an oxypropylene unit, EO is an oxyethylene unit, y is a number
in the range of 0 to about 20, z is a number in the range of 0 to about
20, the sum y+z is a number in the range of about 2 to about 20, and
R.sub.2 and R.sub.3 are selected, independently, from the group consisting
of hydrogen, an alkyl group, an aralkyl group and an aryl group;
R.sub.1 is either methylene or oxygen;
n is a number in the range of one to about 8; and
E is hydrogen or methylol when R.sub.1 is methylene, or E is hydroxy when
R.sub.1 is oxygen; and (d) a suitable carrier comprising water.
Another important aspect of the present invention is to provide an
indicator reagent composition capable of interacting with albumin and
undergoing a color change and including a hydrophobic polymeric compound
of general structural formula (I) wherein R.sub.2 and R.sub.3 are
selected, independently, from the group consisting of hydrogen, an alkyl
group including from one to about 22 carbon atoms, .alpha.-methylstyryl
and phenyl; or wherein the moiety --A--R.sub.1 --of the hydrophobic
polymeric compound of general structural formula (I) is selected from the
group consisting of
##STR7##
wherein R.sub.2 ' and R.sub.3 ' are, independently, hydrogen or an alkyl
group including from one to about 22 carbon atoms.
Another important aspect of the present invention is to provide an
indicator reagent composition for the assay of protein that includes a
hydrophobic polymeric compound of general structural formula (I), wherein
n is a number in the range of about 2 to about 5, and/or wherein the
moiety --A--R.sub.1 --of the hydrophobic polymeric compound is
##STR8##
wherein y' and z', independently, are numbers in the range of about 2 to
about 8, and especially about 5 to about 6; the sum y'+z' is a number in
the range of about 6 to about 16, and especially about 10 to about 12; and
R.sub.2 ' is an alkyl group, linear or branched, including from about 6 to
about 18, and especially from about 7 to about 12, carbon atoms. To
achieve the full advantage of the present invention, R.sub.2 ' is an alkyl
group including from about 8 to about 10 carbon atoms, like the C.sub.9
H.sub.19 alkyl group.
Another aspect of the present invention is to provide a method of assaying
for protein by incorporating an indicator reagent composition, including a
hydrophobic polymeric compound of general structural formula (I), into a
dry phase analyte detection device.
Still another aspect of the present invention is to provide a new and
improved method of assaying for protein by utilizing an analyte test
device including a carrier matrix having incorporated therein an indicator
reagent composition capable of interacting with the protein content in a
test sample, wherein the carrier matrix comprises a bibulous matrix, like
filter paper, or a non-bibulous matrix, like a layer, film or membrane of
permeable polymeric material.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and other aspects and advantages and novel features of the
present invention will become apparent from the following detailed
description of the preferred embodiments of the invention illustrated in
the accompanying figure demonstrating the increased sensitivity of test
strips including an indicator reagent composition of the present invention
to proteins, thereby permitting more accurate and differentiable analyte
determinations:
FIG. 1 is a dose response plot for albumin concentration (in mg/dL) vs. the
Kubelka-Munk function (K/S) for reflectance at 630 nm (nanometers)
comparing test strips incorporating an indicator reagent composition
including a hydrophobic polymeric compound of general structural formula
(I) to test strips incorporating an indicator reagent composition absent a
hydrophilic polymeric compound.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, the qualitative or quantitative
assay for proteins, such as albumin, and especially for negative to trace
concentrations of proteins, in urine and other liquid test samples is
accomplished by utilizing an indicator reagent composition including: (a)
an indicator dye, (b) a buffer, (c) a hydrophobic polymeric compound
depicted by general structural formula (I):
##STR9##
wherein A is
##STR10##
and PO is an oxypropylene unit, EO is an oxyethylene unit, y is a number
in the range of 0 to about 20, z is a number in the range of 0 to about
20, the sum y+z is a number in the range of about 2 to about 20, and
R.sub.2 and R.sub.3 are selected, independently, from the group consisting
of hydrogen, an alkyl group, an aralkyl group and an aryl group;
R.sub.1 is either methylene or oxygen;
n is a number in the range of one to about 8; and
E is hydrogen or methylol when R.sub.1 is methylene, or E is hydroxy when
R.sub.1 is oxygen, and (d) a suitable carrier comprising water.
Preferably, R.sub.2 and R.sub.3 are, independently, hydrogen, a linear or
branched alkyl group including from one to about 22 carbon atoms,
.alpha.-methylstyryl or phenyl. In addition, the moiety --A--R.sub.1 --is
preferably
##STR11##
wherein R.sub.2 ' and R.sub.3 ' are, independently, hydrogen or an alkyl
group. To achieve the full advantage of the present invention, the
hydrophobic polymeric compound of general structural formula (I) includes
a moiety --A--R.sub.1 --having the structure:
##STR12##
wherein y' and z' are, independently, numbers in the range of about 2 to
about 8, and preferably about 5 to about 6; the sum y'+z' is a number in
the range of about 6 to about 16, and preferably about 10 to about 12; and
R.sub.2 ' is an alkyl group, linear or branched, including from about 6 to
about 18 carbon, and preferably from about 7 to about 12 carbon atoms;
and/or n is a number in the range of about 2 to about 5. In an especially
useful embodiment, R.sub.2 ' is a nonyl alkyl group (C.sub.9 H.sub.19).
By employing an indicator reagent composition including a hydrophilic
polymeric compound of general structural formula (I), the assay achieves a
sufficient sensitivity to proteins and sufficient visual color resolution
between protein levels to permit the differentiation between negative and
trace concentration levels of proteins in liquid test samples. The
improved sensitivity and color resolution to low protein levels afforded
by the method of the present invention is especially useful in urine
assays of test samples including from 0 to about 30 mg/dL protein because
the nun%her of false positive assays for a trace amount of protein in
urine is substantially reduced.
Present-day commercial assays do not effectively differentiate between
protein concentrations in the range of 0 mg/dL to about 30 mg/dL, and
especially between a test sample including less than about 15 mg/dL
protein (a negative assay) and a test sample including from about 15 to
about 30 mg/dL protein (a trace amount of protein). Differentiating
between low protein concentration levels is important in the art because
the range of 0 mg/dL to about 15 mg/dL is regarded as the normal urine
protein level for a healthy individual. Therefore, urine protein levels of
0 mg/dL to about 15 mg/dL is a clinically negative result. A urine protein
level greater than about 15 mg/dL, and up to about 30 mg/dL, is a
clinically significant trace amount of protein that can signify a diseased
state. Upon discovering a trace amount of urinary protein in a screening
assay, the assay is confirmed by a different assay method.
Accordingly, to avoid performing unnecessary, and costly, confirmatory
assays, it is important to provide an assay for urinary proteins that
minimizes the number of false positive assays. It also should be noted
that in regard to urine protein concentrations in the relatively high
range, such as from about 30 mg/dL to about 2000 mg/dL, the method of the
present invention still affords improved sensitivity and color resolution
to urine protein concentration, however such clinical benefits are less
critical in this concentration range since such high protein levels are
definitely indicative of an abnormal physiological state that must be
investigated further.
Presently, urine samples are screened for the presence of excess protein by
contacting a dry phase test strip with the urine. The test strip includes
a test pad having incorporated therein an indicator composition that
undergoes a detectable change in response to the concentration of protein
in the test sample. A common test pad incorporates an indicator
composition including an octahalosulphophthalein indicator dye, a buffer
and optional ingredients such as background dyes, surfactants and color
stabilizers.
This common test strip detects only albumin. However, the test strip
provides an excellent total urinary protein assay because albumin
excretion correlates to total protein excretion. The coffeepot test strips
are packaged with a reference chart having six standard color blocks
determined from standardized albumin samples. The color chart corresponds
to different protein concentrations and provides a rapid assay for urinary
protein. The color chart provides six concentration ranges for urinary
protein; the clinically normal range of negative (less than 15 mg/dL), the
trace range (about 15 to about 30 mg/dL), and the pathological ranges of
30, 100, 300 and 2000 mg/dL. The color change produced over the entire
range is yellow to yellowish-green to green to blue. For the negative
range to trace range, the color change is from a greenish-yellow to a
light green. Such a color change is difficult for an assayer to
differentiate visually.
If the color change is interpreted as showing a trace amount of protein in
the urine, a confirmatory assay of the urine is required. Accordingly, if
the test strip provides a large number of false positive assays because of
a poor color differentiation between a negative assay and a trace assay, a
large number of time consuming and costly confirmatory assays would be
performed needlessly. However, it has been difficult to provide a method
and composition that affords a definite color differentiation between a
negative assay and a trace assay, especially because a negative assay
correlates to a normal amount of protein in the urine and because protein
concentration is greater in urine having a high specific gravity.
For urine samples including a high protein concentration, e.g., about 100
mg/dL or greater, the color change for high specific gravity and low
specific gravity urine samples are essentially identical. However, a
specific gravity effect is observed in assaying urine samples including up
to about 30 mg/dL of protein. For example, a high specific gravity urine
sample including a clinically negative amount of protein provides a color
change that closely matches the color change of a low specific gravity
urine sample including a clinically significant trace amount of protein.
Therefore, the assay of the negative high specific gravity urine can be
interpreted, incorrectly, as including a clinically significant trace
amount of protein. Then, the high specific gravity urine will be assayed
by a confirmatory assay method, such as the SSA method, and the
confirmatory assay will fail to find a clinically significant amount of
protein in the urine. Therefore, the false positive screening test for
albumin caused an unnecessary confirmatory assay to be performed.
The color displacement of an assay of a high specific gravity (SG) urine
sample including a negative amount of protein toward the color exhibited
by an assay of a sample including a trace amount of protein is referred to
as a positive SG interference. This interference is the source of the
false positive readings provided by present day test strips. It has been
theorized that the positive interference attributed to a high SG urine
sample is caused by at least two factors, i.e., the presence of quaternary
ammonium compounds in the test sample and the buffering capacity of the
test samples. Quaternary ammonium compounds include normal protein,
peptides, amino acids and creatinine. All of these urinary components
increase in concentration with increased specific gravity. Another factor
that can cause positive SG interference is the buffering capacity of high
SG urine samples, especially phosphate that shifts the pH upward by 0.1
unit.
In addition, a high SG urine sample shows a decreased reactivity to the
indicator reagent composition at the positive protein levels of trace and
above. Dose response plots for urinary protein assays consistently display
a lower slope for high SG urine samples than for low SG urine samples.
Hence, the differentiation between different color blocks becomes more
difficult as SG increases, and is referred to as the negative SG
interference. This decrease in color differentiation, especially in the
trace to 30 mg/dL concentration range, provides a false negative assay.
Therefore, the assayer is aware only of the false positive assays, since
only positive results are confirmed. The assayer is unaware of false
negative test strip readings. Therefore, it is necessary to eliminate the
specific gravity of the urine test sample as a parameter in assaying urine
samples in order to achieve an accurate protein assay in the range of 0 to
about 30 mg/dL.
Furthermore, it will become apparent that in addition to assaying urine,
the method and composition of the present invention also can be used to
determine the presence or quantitative concentration of albumin in blood
plasma or serum; and more generally, the albumin content of many other
albumin-containing fluids as well. In accordance with another important
feature of the present invention, the method and composition of the
present invention is employed in dry phase, test pad assays to determine
the presence or concentration of proteins, especially negative to trace
concentrations of proteins, in urine or other liquid test samples.
Surprisingly and unexpectedly, an indicator reagent composition including a
hydrophobic polymeric compound of general structural formula (I)
demonstrated improved sensitivity and visual color resolution to negative
to trace protein concentrations when used in a dye-binding technique to
determine the presence or concentration of proteins in a test sample. The
dye-binding technique using the hydrophobic polymeric compound of general
structural formula (I) in the indicator reagent composition provides a
more accurate, trustworthy and clinically significant assay especially for
negative to trace concentrations of protein.
A test pad comprising an indicator reagent composition of the present
invention incorporated into a suitable carrier matrix has demonstrated
improved color resolution and increased sensitivity to low protein
concentrations when used in a dye-binding technique to determine the
presence or concentration of proteins in a test sample. The dye-binding
technique using an indicator reagent composition of the present invention
incorporated into a suitable carrier matrix provides a more accurate,
trustworthy and clinically significant quantitative assay for protein in
the range of 0 to about 30 mg/dL.
The indicator reagent compositions used in present day assay methods for
protein interact with proteins and undergo a color transition due to the
protein-error phenomena when maintained at the proper, constant pH. The
protein-error phenomena is fully described in Free et al. U.S. Pat. No.
3,095,277; Atkinson et al. U.S. Pat. No. 3,438,737; and Keston U.S. Pat.
No. 3,485,587, wherein the various dyes, the correct pH ranges, the
buffers and the carrier matrices, such as bibulous substrates, like filter
paper, required to observe the protein-error phenomena are disclosed. The
three above-identified patents basically describe the present day, dry
phase test strips employed to assay for total protein content in urine.
These total protein test strips generally include an indicator reagent
composition comprising an indicator dye that normally undergoes a color
transition at a strongly acidic pH of 5 or below and a buffer to maintain
the pH of the indicator dye slightly below the pH of color transition for
the dye. A sufficient buffering of the indicator dye essentially assures
that the dye changes color due to an interaction with protein rather than
due to a pH change occurring upon contact with the test sample. The
present day total protein test strips further include a carrier matrix,
such as untreated or treated filter paper, for incorporation of the
indicator reagent composition.
In accordance with an important feature of the present invention, the
carrier matrix incorporates an indicator reagent composition of the
present invention including a suitable indicator dye. A suitable indicator
dye is capable of interacting with proteins, and is capable of undergoing
a sufficient color transition due to the protein-error phenomena upon
interaction with a protein to give a detectable or measurable response.
However, in accordance with the present invention, it has been found that
incorporating a suitable hydrophobic polymeric compound of general
structural formula (I) into the indicator reagent composition
substantially improves the color resolution and differentiation, both
visually and instrumentally, of the color transition occurring upon
interaction of the indicator dye with protein, especially when the test
sample includes from 0 to about 30 mg/dL of protein. Therefore, the
sensitivity of the protein assay, especially an low protein concentrations
in the negative to trace range, is increased, and the number of false
positive assays for urinary protein is substantially reduced.
The method of the present invention utilizes the "protein-error" phenomena
previously discussed. However, the incorporation of a suitable hydrophobic
polymeric compound of general structural formula (I) into the indicator
reagent composition of the present invention improves the color resolution
and differentiation of the color transition occurring because of the
dye-protein interaction. As previously described, when a pH indicator dye
interacts with a protein, the apparent pK.sub.a of the dye is altered and
a color transition occurs producing the so-called "protein-error"
phenomenon. A slight color transition, i.e., a background color, occurs
even if the test sample includes a normal amount of protein, i.e., assays
as negative. This slight color transition is sufficient to cause an
incorrect interpretation of a negative assay as a trace assay. The
development of a background color is attributed in most part to the
reactivity of the indicator dye toward a positive SG interference.
However, by employing the indicator reagent composition of the present
invention, color development is essentially independent of test sample
specific gravity. Consequently, color resolution and color differentiation
upon interaction of the indicator dye with proteins is improved, assay
sensitivity is increased, and the number of false positive assays is
substantially reduced.
It has been demonstrated that the indicator reagent composition of the
present invention provides an improved protein assay for an
albumin-containing sample, and especially a sample containing 30 mg/dL or
less protein, by essentially eliminating the positive SG interference.
Thus, by essentially eliminating the positive SG interference, a negative
assay for protein is maintained in the yellow region of the color space.
The color resolution and color differentiation of the color transition
resulting from assaying a test sample including 30 mg/dL or less albumin
therefore are improved.
In general, any pH indicator dye can be utilized in the composition of the
present invention, provided that the dye is capable of interacting with
proteins and of undergoing a detectable and measurable color transition in
response to the protein interaction. Such indicator dyes are well-known in
the art and are utilized in indicator reagent compositions in methods to
determine the presence or the concentration of protein in urine or other
liquid test samples. In addition to the indicator dyes, it is known that
the indicator reagent composition also may require a sufficient amount of
a proper buffer, such that the indicator dye does not change color as a
result of a pH shift, but changes color upon contact and interaction with
proteins to accurately establish the presence or concentration of protein
in the test sample. Further, it has been demonstrated that any of various
known types of buffers can be used in the indicator reagent composition.
For optimum results, it also has been found that the pH of the indicator
reagent composition generally should be maintained at a pH value only
slightly below the pH range wherein the indicator dye of the indicator
reagent composition undergoes a color transition. A method of determining
a suitable buffered pH value for the particular indicator dye of the
indicator reagent composition and of determining the particular buffer
that can be used in the indicator reagent composition is found in Keston,
U.S. Pat. No. 3,485,587.
Furthermore, the indicator dye employed in the indicator reagent
composition undergoes a sufficiently intense color transition such that
relatively low concentrations of protein in the test sample produces a
detectable and measurable color transition. Therefore, in order to achieve
the full advantage of the present invention, the indicator dyes employed
in the indicator reagent composition are selected such that the dye
undergoes a sufficient color change either from a more intense color to a
less intense color, or from a less intense color to a more intense color,
such that the assayer, either visually or by instrument, can detect and
measure the protein content of the test sample.
It has been found that the indicator dye used most advantageously in the
composition and method of the present invention is an
octahalosulfophthalein-type or an octahalophenolphthalein-type dye, such
as tetrabromophenol blue, tetrachlorophenol blue,
3',3",5',5"-tetraiodo-3,4,5,6-tetrabromophenolsulfophthalein and
3,3"-diiodo-5,5",3,4,5,6-hexabromophenolsulfophthalein. These
octahalosulfophthalein-type and octahalophenolphthalein-type dyes can
undergo a sufficient color transition after binding to a protein to allow
the visual or instrumental detection and measurement of the protein
content of a test sample, including a negative to trace concentration of
protein in the sample.
Depending upon several chemical and physical parameters, such as ability to
interact with proteins, color of the test sample, intensity of the color
transition and chemical compatibilities, a particular
octahalosulfophthalein-type dye or octahalophenolphthalein-type dye is
selected as the indicator dye of the indicator composition. The indicator
dye generally is present in the indicator reagent composition in an amount
of about 0.05 to about 0.6 millimoles per liter of the composition.
However, the indicator dye can be present in a greater or lesser amount
depending upon the intensity of the color transition of the particular
indicator dye that is used. The exact octahalosulfophthalein-type dye or
octahalophenolphthalein-type dye selected as the indicator dye of the
indicator reagent composition can be determined by those skilled in the
art of designing test kits in order to produce an assay for proteins
having maximum visual color resolution and maximum sensitivity. The
octahalosulfophthalein-type dye or octahalophenolphthalein-type dye
utilized in the indicator reagent composition of the present invention can
be prepared by methods well known to person in the art. Furthermore,
several dye compounds that are useful in the method of the present
invention are well known indicator dyes that are presently available
commercially.
Further, it has been demonstrated that any of various known types of
buffers can be used in the indicator reagent composition of the present
invention. The function of the buffer is to maintain the composition at a
substantially constant pH to produce the desired color change in the
indicator reagent composition because of the presence of albumin and to
essentially eliminate color changes due to a variation in the pH of the
albumin-containing test sample. As a result, the amount of buffer used
depends upon the nature of the test sample. The quantity of buffer usually
falls between about 250 millimolar and 750 millimolar (millimoles per
liter), although in particular cases the quantity of buffer can be above
or below this range.
The nature of the particular buffer used will depend upon, and vary with,
the indicator dye incorporated into the indicator reagent composition.
However, it generally has been found that for optimum results, the pH of
the indicator reagent composition should be maintained at a value only
slightly below the pH range wherein the indicator dye undergoes a color
transition. Useful buffers include, for example, but are not limited to,
citric acid, maleic acid, tartaric acid, phthalic acid, sulfosalicylic
acid, succinic acid, maleic acid, and malonic acid; their respective
alkali metal and ammonium salts; and other suitable buffers are as well
known in the art, or combinations thereof. A method of determining the
proper pH for the particular indicator dye and the particular buffer used
in the composition can be found in Keston, U.S. Pat. No. 3,485,587.
In addition to the indicator dye and the buffer, the indicator reagent
composition includes a hydrophobic polymeric material having the general
structural formula (I):
##STR13##
wherein A is
##STR14##
and PO is an oxypropylene unit, EO is an oxyethylene unit, y is a nun%her
in the range of 0 to about 20, z is a number in the range of 0 to about
20, the sum y+z is a number in the range of about 2 to about 20, and
R.sub.2 and R.sub.3 are selected, independently, from the group consisting
of hydrogen, an alkyl group, an aralkyl group, and an aryl group;
R.sub.1 is either methylene or oxygen;
n is a number in the range of one to about 8; and
E is hydrogen or methylol when R.sub.1 is methylene, or E is hydroxy when
R.sub.1 is oxygen. The hydrophobic polymeric compound of general
structural formula (I) has a molecular weight of about 800 to about
12,000, and preferably of about 1500 to about 8000. It has been found that
a hydrophobic polymeric compound depicted by general structural formula
(I) reduces the development of a background color; reduces positive
interferences attributed to urinary components other than proteins; and
substantially reduces the positive interferences attributed to high
specific gravity samples that, in turn, lead to false positive assays in
dry phase test strip assays.
As will be discussed more fully hereinafter, it has been shown that about
1% to about 8%, and preferably about 2% to about 6%, by weight of a
hydrophobic polymeric compound of general structural formula (I), per
milliliter of indicator reagent composition, provides a test pad that
remains a light yellow color after contact with a high SG (specific
gravity) or a low SG urine sample that is free of albumin. In contrast, a
test pad incorporating a reagent composition lacking the hydrophobic
polymeric compound of general structural formula (I) changes to a
greenish-yellow color after contact with a high SG urine sample free of
albumin. This color transition from yellow to greenish-yellow can be
interpreted, incorrectly, as a clinically significant trace amount of
albumin and therefore provide a false positive assay. The test sample then
would be subjected to an unnecessary and costly confirmatory assay for
albumin.
With regard to a hydrophobic polymeric compound of general structural
formula (I), preferably, R.sub.2 and R.sub.3 are, independently, hydrogen,
a linear or branched alkyl group including from one to about 22 carbon
atoms, .alpha.-methylstyryl or phenyl. In addition, the moiety
--A--R.sub.1 --preferably is
##STR15##
wherein R.sub.2 ' and R.sub.3 ' are, independently, hydrogen or an alkyl
group including one to about 22 carbon atoms. To achieve the full
advantage of the present invention, the hydrophobic polymeric compound of
general structural formula (I) includes the number n in the range of about
2 to about 5 and/or includes a moiety --A--R.sub.1 --having the structure:
##STR16##
wherein y' and z' are, independently, numbers in the range of about 2 to
about 8, and preferably from about 5 to about 6; the sum y'+z' is a number
in the range of about 6 to about 16, and preferably about 10 to about 12;
and R.sub.2 ' is an alkyl group, linear or branched, including from about
6 to about 18, and preferably from about 7 to about 12, carbon atoms.
A particularly useful hydrophobic polymeric compound of general structural
formula (I) is depicted in structural formula (II), referred to
hereinafter as polymer II. Polymer II has a molecular weight of about
2200.
##STR17##
In addition to the essential ingredients, other optional ingredients that
do not materially alter the nature or the function of the essential
ingredients, and that do not interfere with the assay for proteins, also
can be included in the indicator reagent composition. For example, the
indicator reagent composition optionally can include a compound to improve
test sample wetting of the test pad of the test device. The optional
wetting compound usually is a nonionic surfactant. The octoxynols,
nonoxynols and ethoxylated fatty alcohols are nonlimiting examples of
nonionic surfactants useful as the wetting compound in the indicator
reagent composition of the present invention. The wetting compound is
included in the indicator reagent composition in a concentration of 0 nM
to about 200 mM, and preferably in a concentration of from about 50 nM to
about 200 mM.
In addition, to improve the color resolution and differentiation of the
color transition in a chromogenic assay for proteins, inert background
dyes can be included in the indicator reagent composition. Suitable
background dyes include, but are not limited to, ethyl orange
(4-(4-diethylaminophenylazo)benzenesulfonic acid); Orange G
(4-(2-hydroxy-(7,9 sodium disulfonate)-1-naphthylazo)benzene); disperse
orange 11, 13, or 25; calcomine orange; methyl orange; and orange II
(4-(2-hydroxy-1-naphthylazo)benzenesulfonic acid), or combinations
thereof. A background dye is included in the indicator reagent composition
of the present invention in a concentration of 0 mM to about 2 mM, and
preferably about 0.1 mM to about 1 mM.
The carrier vehicle for the ingredients included in the indicator reagent
composition includes water. However, because of the limited water
solubility of particular ingredients included in the indicator reagent
composition, organic solvents such as methanol, ethanol, isopropyl
alcohol, ethylene glycol, propylene glycol, acetone, dimethylformamide,
dimethylsulfoxide, acetonitrile, ethyl acetate and similar solvents can be
used to solubilize the water-insoluble ingredients. The selection of a
suitable organic solvent or combination of organic solvents to include in
the carrier vehicle of the indicator reagent composition is within the
capability of those skilled in the art of designing diagnostic test
strips.
Furthermore, it should be understood that the indicator reagent composition
can be composed of two distinct solutions, one aqueous-based and one
organic solvent-based, to solubilize the water soluble and the water
insoluble components respectively. In such a case, the amount of each
ingredient present in the composition is determined using the total volume
of the two distinct solutions. As will be demonstrated more fully
hereinafter, the carrier matrix then can be subjected to two treatments.
Accordingly, a first solution, either the aqueous or the organic
solvent-based solution, is incorporated into the carrier matrix, then the
second solution is incorporated into the carrier matrix in order to
homogeneously incorporate each essential ingredient of the indicator
reagent composition into the carrier matrix.
Upon contact with the urine or other test sample, a color transition of the
indicator reagent composition demonstrates the presence of protein.
Furthermore, the intensity and degree of the color transition can be used
to determine the quantitative concentration of protein in the test sample
by comparing or correlating the color produced by the test sample to
colors produced by solutions having a known concentration of protein. In
accordance with an important feature of the present invention, it has been
demonstrated that the indicator reagent composition provides a
sufficiently resolved and differentiated color transition such that the
amount of protein, including negative to trace amounts of protein, in the
test sample can be measured and accurately determined without the use of
color-measuring instruments, such as spectrophotometers or colorimeters.
However, if desired, such color-measuring instruments can be used to
measure the difference in color degree and intensity between the test
sample and a solution of known albumin concentration. In addition, it has
been demonstrated that the assay is essentially independent of urine
specific gravity, thereby substantially reducing the number of false
positive assays.
Accordingly, an assay for protein that utilizes an indicator reagent
composition of the present invention including a hydrophobic polymeric
compound of general structural formula (I) improves the accuracy and
reliability of the assay and also increases physician confidence in the
assay. Additionally, because of the number of urine assays for protein
being performed at home by the untrained patient, as opposed to trained
physicians or technicians in the laboratory, it is imperative to provide
accurate and reliable quantitative assay methods for negative to trace
protein content in a urine sample to reduce the number of false positive
assays resulting from urine specific gravity.
In general, assays for protein are conducted at an acidic pH using an
indicator dye undergoing a color transition at an acidic pH because the
indicator dye can interact more strongly with the protein at low, acidic
pH values. The increased interaction between the indicator dye and the
protein at low pH values occurs because of a strong attraction between the
positively-charged cationic protein molecule and the negatively-charged
anionic indicator dye molecule, and, additionally, because the acidic
conditions serve to partially denature proteins and therefore increase the
ability of the protein to interact with the indicator dye. Therefore, the
indicator reagent composition of the present invention generally is
adjusted to and maintained at an acidic pH. Generally, the pH of the
system is adjusted to and maintained at between about 2 and about 4; and
to achieve the full advantage of the present invention the pH is adjusted
to and maintained at between about 3 and 4.
To demonstrate the new and unexpected results achieved by the method and
composition of the present invention, an indicator reagent composition,
including a hydrophobic polymeric compound of general structural formula
(I), was prepared, then used in a dry phase test strip assay for the total
protein content of a test sample. The indicator dye, tetrabromophenol blue
(TBPB), interacts with proteins and undergo a color transition at a pH of
about 3.5. The TBPB is yellow in color in the absence of proteins and
changes color ranging from yellowish-green, to green to blue in the
presence of increasing amounts of proteins. As a result, an indicator
reagent composition including the appropriate amount of TBPB, adjusted to
and maintained at a pH of 3.5 with a suitable buffer, produced the color
transitions summarized in TABLE I upon contact with standardized protein
solutions.
TABLE I
______________________________________
COLOR TRANSITION OF TBPB-CONTAINING
INDICATOR REAGENT COMPOSITION UPON
INTERACTION WITH STANDARDIZED PROTEIN
SOLUTIONS (pH = 3.5)
Concentration of Standardized
Protein Solution (mg/dL)
Observed Color
______________________________________
0-15 (negative) yellow
15-30 (trace) yellowish green
30 light green
100 medium green
300 blue green
2000 dark blue green
______________________________________
The dry phase, test pad assay for albumin utilizing an indicator reagent
composition of the present invention is performed in accordance with
methods well known in the art. In general, the albumin assay is performed
by contacting an analyte detection device with the urine or other test
sample. The analyte detection device comprises a test pad incorporating
the indicator reagent composition. The analyte detection device can be
dipped into the urine or serum sample, or the urine or serum sample can be
applied to the analyte detection device dropwise. A change in color of the
test pad of the device demonstrates the presence of albumin, and, if so
designed, the intensity and depth of the color change can be compared to a
color chart to afford a quantitative measurement of the concentration of
albumin in the test sample.
Typically, the analyte detection device is a test strip impregnated with a
reagent composition, designed either as a single pad test strip (to assay
for a single analyte) or as a multiple pad test strip (to assay for
several analytes simultaneously). For either type of reagent impregnated
test strip, the test strip includes a support strip, or handle, normally
constructed from a hydrophobic plastic, and a reagent test pad, comprising
a bibulous or nonbibulous carrier matrix. In general, the carrier matrix
is an absorbent material that allows the test sample to move, in response
to capillary forces, through the matrix to contact the reagent composition
and produce a detectable and measurable color transition.
The carrier matrix can be any substance capable of incorporating the
chemical reagents required to perform the assay of interest, as long as
the carrier matrix is substantially inert with respect to the chemical
reagents. The carrier matrix also is porous or absorbent relative to the
liquid test sample.
The expression "carrier matrix" refers to either bibulous or nonbibulous
matrices that are insoluble in water and other physiological fluids and
maintain their structural integrity when exposed to water and other
physiological fluids. Suitable bibulous matrices include filter paper,
sponge materials, cellulose, wood, woven and nonwoven fabrics and the
like. Nonbibulous matrices include glass fiber, polymeric films, and
preformed or microporous membranes. Other suitable carrier matrices
include hydrophilic inorganic powders, such as silica gel, alumina,
diatomaceous earth and the like; argillaceous substances; cloth;
hydrophilic natural polymeric materials, particularly cellulosic material,
like cellulosic beads, and especially fiber-containing papers such as
filter paper or chromatographic paper; synthetic or modified
naturally-occurring polymers, such as cellulose acetate, polyvinyl
chloride, polyacrylamide, polyacrylates, polyurethanes, crosslinked
dextran, agarose, and other such crosslinked and noncrosslinked
water-insoluble hydrophilic polymers. Non-absorptive substances are not
suitable for use as the carrier matrix of the present invention. However,
a hard, porous plastic is useful as the carrier matrix as long as the
plastic is sufficiently porous to allow the test sample to permeate
through the plastic and contact the indicator reagent composition. The
carrier matrix can be of different chemical compositions or a mixture of
chemical compositions. The matrix also can vary in regards to smoothness
and roughness combined with hardness and softness. The handle usually is
formed from hydrophobic materials such as cellulose acetate, polyethylene
terephthalate, polycarbonate or polystyrene, and the carrier matrix is
most advantageously constructed from bibulous filter paper or nonbibulous
permeable polymeric films.
To achieve the full advantage of the present invention, the indicator
reagent composition is homogeneously incorporated into a suitable carrier
matrix and utilized in a dry phase test strip for the assay or protein in
a test sample. The method of the present invention affords an economical,
accurate and reliable assay for the total concentration of protein in test
samples that can be performed at home or in the laboratory. In addition,
the method of the present invention is essentially independent of test
sample specific gravity, and allows detection of, and differentiation
between, negative and trace protein concentrations in a test sample
thereby making the assay more useful clinically.
In accordance with the method of the present invention, to perform a dry
phase, test strip assay for protein, an aqueous solution, including from
about 250 mM (millimolar, or millimoles per liter) to about 750 mM total
concentration of a buffer, such as potassium citrate, adjusted to a pH of
about 3.5, first is prepared. A bibulous matrix, such as filter paper,
like WHATMAN CCP500 filter paper, available commercially from Whatman
Ltd., Maidstone, Kent, U.K., then is saturated with the aqueous solution
containing the buffer either by spreading, by immersing or by spraying the
aqueous solution onto sheets or precut strips of the filter paper. The
aqueous solvent is removed from the filter paper by oven drying in an air
oven at about 50.degree. C. for about 20 minutes.
Then, a tetrahydrofuran (THF) or ethanol solution, including about 0.05 to
about 0.6 mM of an indicator dye, like about 0.3 mM of TBPB, and from
about 1% to about 8%, by weight volume, of a hydrophobic polymeric
compound of general structural formula (I), like 5% (w/v) of polymer II,
is prepared. The strip of dried filter paper incorporating the citrate
buffer then is saturated with the THF or ethanol solution including the
indicator dye and the polymer II. After removing the THF or ethanol
solvent by oven drying at about 50.degree. C. for about 15 to about 30
minutes, the reagent impregnated filter paper strip is cut to an
appropriate size, such as a pad having dimensions from about 0.25 cm by
about 0.5 cm to about 0.5 cm by about 1.0 cm. The reagent impregnated
filter paper pad then is secured to a plastic handle with double sided
adhesive to provide a test strip.
The test strip then was dipped into a fresh, uncentrifuged urine sample for
a sufficient time to saturate the test pad with the sample. The test strip
should not be immersed in the urine sample for longer than about 3 to 5
seconds in order to avoid extraction of the buffer from the filter paper
by the urine sample. After removing the test strip from the urine sample
and waiting a predetermined time, such as from about 1 minute to about 2
minutes, the test strip is examined, either visually or by instrument, for
a response. The color transition, if any, of the test pad reveals the
presence and/or concentration of protein in the urine sample.
It is well within the experimental techniques of those skilled in the art
of preparing test devices to determine the proper balance between size of
reagent pad, the strength of the indicator reagent composition, the amount
of test sample, and the method of introducing the test sample to the test
strip, such as by piperting rather than dipping, in order to design a
quantitative assay for protein utilizing the method and composition the
present invention.
In many cases simple visual observation of the test strip provides the
desired information. If more accurate information is required, a color
chart bearing color spots corresponding to various know protein
concentrations, can be prepared for the particular indicator reagent
composition incorporated into the test strip. The resulting color of the
test strip after contact with the urine sample then can be compared with
the color spots on the chart to determine the protein concentration of the
test sample.
If a still more accurate determination is required, a spectrophotometer or
colorimeter can be used to more precisely determine the degree of color
transition. In addition, the dry phase test strip assay can be made
quantitative by employing spectrophotometric or colorimetric techniques,
as opposed to visual techniques, in order to more reliably and more
accurately measure the degree of color transition, and therefore more
accurately measure the concentration of protein in the test sample,
especially at lower protein concentrations, such from 0 mg/dL to about 30
mg/dL.
As will be discussed more fully hereinafter, the ability to detect and
differentiate between negative and trace concentrations of protein in a
test sample by employing an indicator reagent composition of the present
invention surprisingly and unexpectedly provides an improved method of
assaying for the total protein content of liquid test samples. For
example, according to present day methods, differentiation between an
assay indicating a clinically significant trace amount of protein (e.g.,
about 15 to about 30 mg/dL protein) and a negative assay (e.g., about 15
mg/dL protein or less) is difficult, thereby resulting in a large number
of false positive assays.
A major cause of false positive assays is the dependence of the protein
assay on test sample specific gravity. Accordingly, until the method of
the present invention, no dry phase test strip technique either was
essentially independent of test sample specific gravity or was available
to consistently differentiate between the negative and trace
concentrations of protein often found in urine. Therefore, in accordance
with an important feature of the present invention, it has been
demonstrated that by incorporating an indicator reagent composition of the
present invention into a suitable carrier matrix, the presence of a
negative or a trace concentration of protein in a urine sample can be
determined, and differentiated from one another, by using a dry phase test
strip that is essentially independent of the specific gravity of urine
sample.
To show the new and unexpected results achieved by using the indicator
reagent composition of the present invention, test strips incorporating an
indicator reagent composition of the present invention were prepared, and
then were compared to prior art test strips in assays of standardized
urine samples including albumin in the range of 0 mg/dL to about 30 mg/dL.
First, two sets of dry phase test strips were prepared by the two
immersion procedure described above. All the test strips utilized WHATMAN
CCP500 filter paper as the carrier matrix. All the test strips were
immersed into a 0.50M potassium citrate solution buffered at pH 3.5, then
dried. Then, a portion of the test strips, Test Strips A, had incorporated
therein a 0.30 mM solution of TBPB in THF. Test Strips A are prior art
test strips. The remaining test strips incorporating the citrate buffer
had incorporated therein a THF solution that was 0.30 mM in TBPB and
included 5% (w/v) polymer II. These test strips, Test Strips B,
incorporated an indicator reagent composition of the present invention.
Test Strips A were compared to Test Strips B for an ability to detect 0
mg/dL to 30 mg/dL albumin in a urine sample. In particular, a urine pool,
having a low SG of about 1,007 and shown by immunoassay to be free of
albumin, was spiked to various clinically significant albumin levels with
human serum albumin. The spiked urine samples included either 0, 10, 15,
20 or 30 mg/dL of albumin. These standardized urine samples then were used
to compare Test Strips A to Test Strips B for an ability to detect urinary
protein and to differentiate between urinary protein assays in the range
of 0 mg/dL to about 30 mg/dL protein.
Test Strips A and Test Strips B each were immersed into the standardized
urine samples, then examined for a response to the protein content of the
test sample. FIG. 1 illustrates the dose responses to albumin for Test
Strips A and Test Strips B. In particular, FIG. 1 includes two dose
response plots for albumin concentration (mg/dL) rs. K/S at 630 nm
(nanometers). Individual assay results were determined by taking a
reflectance measurement with a reflectance photometer at a suitable time
and wavelength for that particular analyte determination. The reflectance,
as taken from the reflectance scale of zero to one, was incorporated into
the Kubelka-Munk function:
K/S=(1-R).sup.2 /2R,
wherein K is the absorption coefficient, S is the scattering coefficient
and R is reflectance. In FIG. 1, the K/S values were plotted against the
concentration of albumin in the test sample. Generally, it can be stated
that as reflectance decreases, the K/S value increases.
Therefore, the two dose response plots of FIG. 1 show the effects of
increased albumin concentration on the K/S values and the effect of
including a hydrophobic polymeric compound of general structural formula
(I) in the indicator reagent composition. The reflectance was measured at
a wavelength of 630 nm (nanometers), then the K/S values were calculated.
The K/S values are the average K/S values for three replicate
determinations.
From FIG. 1, it is observed that as the albumin concentration increases the
K/S value also increases. Therefore, the reflectance has decreased,
indicating a greater color transition in the test pad. It also is observed
from the dose response plots of FIG. 1 that including a hydrophobic
polymeric compound of general structural formula (I), e.g., polymer II, in
the indicator reagent composition lowers both the intercept and the slope
of the human serum albumin (HSA) dose response plot, as illustrated by the
dashed line of FIG. 1, compared to an indicator reagent composition
lacking the polymer, as illustrated by the solid line of FIG. 1.
It should be noted that the lowering of the intercept from about 0.150 to
about 0.108 by a composition of the present invention is an important and
unexpected result. FIG. 1 shows that the prior art reagent composition
incorporated into Test Strips A and the indicator reagent composition of
the present invention incorporated into Test Strips B each have a
sufficient sensitivity to the amount of albumin in the standardized urine
sample. But the lower intercept provided by the indicator reagent
composition of the present invention incorporated into Test Strips B
maintains the definite yellow color of the test pad after a Test Strip B
contacts a test sample including 0 mg/dL albumin, whereas a Test Strip A
becomes a yellowish-green color after contact with a test sample including
0 mg/dL albumin. For comparative purposes, a K/S value in the range of
about 0.2 to about 0.4 corresponds to a trace amount of protein in the
urine sample. A test pad having a K/S value of about 0.2 is visualized as
a light greenish-yellow color. A trace amount of protein begins at a
protein concentration of about 15 mg/dL.
Accordingly, the yellowish-green color of Test Strip A can be interpreted
incorrectly as including a trace concentration (about 15 mg/dL) of albumin
when the test solution only contains approximately 5 mg/dL albumin.
However, Test Strip B will correctly only turn greenish-yellow at a trace
concentration (about 15 mg/dL) of albumin in a test sample. Therefore, an
indicator reagent composition of the present invention, including a
hydrophobic polymeric compound of general structural formula (I), helps
maintain the color of the test pad in the negative assay range, thereby
substantially reducing the number of false positive assays for a trace
amount of albumin. Consequently, substantially fewer unnecessary
confirmatory assays for trace albumin are conducted.
The Test Strips A and Test Strips B also were compared in the assay of a
high specific gravity (SG=1.023) urine sample that was shown by an
immunoassay to be free of albumin. The assay results for both the low SG
urine sample and the high SG urine sample including no albumin (i.e., a
negative sample), and used to compare a Test Strip A to a Test Strip B,
are summarized below. Both the high SG urine sample and the low SG urine
sample included 0 mg/dL albumin.
______________________________________
K/S at 630 nm (25 seconds), 0 mg/dL Albumin
Test Strip Low SG Sample
High SG Sample
______________________________________
A 0.151 .+-. 0.010
0.199 .+-. 0.013
B 0.097 .+-. 0.002
0.102 .+-. 0.003
______________________________________
From the summarized data, it is observed that a high SG urine sample,
including a negative amount of albumin (0 mg/dL), exhibits a value of
0.199 when a prior art indicator reagent composition is incorporated into
a test pad (Test Strip A). In contrast, a low SG urine sample including a
negative amount of albumin and assayed by a prior art indicator reagent
composition exhibits a value of 0.151. As discussed above, the 0.151 value
of the low SG urine sample appears as a greenish-yellow color for the
blank reaction that can be interpreted incorrectly as a trace amount of
protein. Accordingly, the value of 0.199 exhibited by the high SG urine
sample provides a greenish-yellow color for the blank reaction that is
displaced even further into the green color region, and thereby is more
easily interpreted incorrectly as a clinically significant positive assay
for a trace amount of protein.
Therefore, the prior art compositions used in assays for protein provided
an unacceptably large number of false positive assays because the color
change of the test strip is directly related to the specific gravity of
test sample. A high SG urine sample showed a significant increase in test
strip reactivity over a low SG urine sample, and accordingly, a greater
number of false positive assays. In contrast, an indicator reagent
composition of the present invention, including a hydrophobic polymeric
compound of general structural formula (I), essentially eliminates
specific gravity as a parameter in the dry phase test strip assay for
proteins.
As previously discussed, a test strip incorporating an indicator reagent
composition of the present invention (Test Strips B) lowers the K/S value
to 0.097 for a low SG urine sample including 0 mg/dL albumin, compared to
a K/S value of 0.151 for test strips incorporating a prior art composition
(Test Strips A). As explained, this reduction in the reactivity provides a
test pad exhibiting a yellow color in the assay of low SG urine samples
including a negative amount of albumin. Surprisingly, a high SG urine
sample including a negative amount of albumin (0 mg/dL) exhibits a K/S
value of 0,102 when assayed by a Test Strip B incorporating a composition
of the present invention. This reactivity also provides a test pad
exhibiting a yellow color that is easily interpreted as a negative assay
for albumin. Therefore, it has been shown that test sample specific
gravity essentially does not effect the color change exhibited by a test
pad incorporating an indicator reagent composition of the present
invention. Consequently, fewer false positive assays for protein result.
The summarized data also shows that a high SG urine sample assayed with a
test strip including a prior art composition exhibits a K/S value of 0.199
and a definite greenish-yellow colored test pad, whereas a high SG urine
sample assayed with a test strip including the present composition
exhibits a K/S value of 0.102 and a yellow colored test pad. Therefore, it
has been shown that the present indicator reagent composition reduces the
reactivity of a test strip in the presence of a high SG urine sample, and
substantially reduces the occurrence of false positive assays due to
interferents present in high SG urine samples.
As a result, it has been demonstrated that using the present indicator
reagent composition to detect the presence and concentration of proteins
in a test sample, surprisingly and unexpectedly allows the detection of,
and differentiation between, a negative and a trace amount of protein in a
test sample. In addition, the assay is essentially independent of, and is
not adversely influenced by, the specific gravity of the test sample. Such
unexpected improvements provide an important and useful advantage over
prior art indicator reagent compositions that lack a hydrophobic polymeric
compound of general structural formula (I), and that are used to assay for
the protein content of test samples. As illustrated in FIG. 1 and in the
data summarized above, the prior methods and compositions suffer from both
a severe specific gravity interference and an inability to effectively
differentiate between a negative albumin concentration of about 15 mg/dL
or less and a clinically significant trace albumin concentration of about
15 to about 30 mg/dL. However, in contrast, an assayer, using a test
device incorporating an indicator composition of the present invention to
assay for albumin in a test sample, can reliably assay test samples for
total protein concentration, including negative and trace amounts of
protein, without generating an unacceptably large number of false positive
assays.
It should be understood that those skilled in the art of designing test
kits are able to design an optimal test strip incorporating a sufficient
amount of a particularly effective indicator reagent composition,
comprising an indicator dye, a buffer and a hydrophobic polymeric compound
of general structural formula (I), to permit the detection of, and the
differentiation between, negative and trace amounts of albumin in a test
sample because assays utilizing the method and composition of the present
invention showed a visually detectable color difference and were
independent of the specific gravity of the test sample. The method and
composition of the present invention allow the assayer to differentiate
between a test sample containing about 15 mg/dL or less of albumin and a
test sample containing about 15 to about 30 mg/dL of albumin, thereby
substantially reducing the number of false positive assays.
Overall, it has been shown that an indicator reagent composition of the
present invention incorporated into a suitable carrier matrix, such as
filter paper, improves the color resolution of assays between test samples
having different protein concentrations and eliminates false positive
results for the total protein content of a liquid test sample, especially
at low protein levels of about 30 mg/dL or less. In addition, the present
composition is not subject to specific gravity interferences. The method
and composition of the present invention also allow visual differentiation
of color transitions resulting from contact of the carrier matrix
impregnated with the present indicator reagent composition with a test
sample containing protein levels of between 0 mg/dL and 30 mg/dL, thereby
providing accurate and trustworthy assays of test samples containing
negative to trace amounts of protein, without generating an unacceptably
high number of false positive assays.
Therefore, in accordance with an important feature of the present
invention, more accurate and reliable assays for total protein content,
and especially for negative or trace total protein content, in urine and
other liquid test samples can be performed by utilizing an indicator
reagent composition including a hydrophobic polymeric compound of general
structural formula (I). The present indicator reagent composition improves
the color resolution of the test strips between test samples having
different protein concentrations, and therefore improves assay
sensitivity, especially at negative to trace albumin levels of
approximately 30 mg/dL and below.
Obviously, many modifications and variations of the invention as
hereinbefore set forth can be made without departing from the spirit and
scope thereof and therefore only such limitations should be imposed as are
indicated by the appended claims.
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